Vol. 29, 2011, No.

6: 616623

Czech J. Food Sci.

Preparation and Characterisation of Food-Grade Chitosan

from Housefly Larvae
Ai-Jun Zhang1,2, Qi-Lian Qin1, Huan Zhang1, Hong-Tuo Wang1, Xuan Li1,
LinMiao1 and Yi-Jun Wu1
1

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute

of Zoology, Chinese Academy of Sciences, Beijing, P.R. China; 2Graduate University of Chinese
Academy of Sciences, Beijing, P.R. China

Abstract
Zhang A.-J., Qin Q.-L., Zhang H., Wang H.-T., Li X., Miao L., Wu Y.-J. (2011): Preparation and characterisation of food-grade chitosan from housefly larvae. Czech J. Food Sci., 29: 616623.
The preparation and characterisation of food-grade chitosan from housefly larvae are reported. A refinement procedure
was developed to remove larval mouth hooks from the primary chitosan product, which greatly improved the quality
of the final product and simplified the production procedures. Different factors affecting chitosan preparation were
studied and an orthogonal experiment was designed to determine optimal preparation conditions. When prepared under optimal reaction conditions, the end product was snow-white in colour, had a high deacetylation percentage, good
viscosity, and a low ash content. The end product was characterised by Fourier transform infrared spectral analysis,
X-ray diffraction analysis, thermo-gravimetric analysis, and differential scanning calorimetry. Its physical and chemical properties and sanitary index were determined and compared to the relevant Chinese standards. The results show
that the chitosan we produced under optimal conditions meets the Chinese Fishery Trade Standard SC/T3403-2004 for
food-grade chitosan.
Keywords: Musca domestica; commercial applications; chitin; insect; derivates

Abbreviations
DDA degree of deacetylation; PSM prortion of the solution to material; RSM ratio of the solution (ml) to material consider changing PSM to RSM, TGA thermo-gravimetric analysis; DSC differential scanning calorimetry;
FTIR Fourier transform infrared; XRD X-ray diffraction

The housefly Musca domestica (Diptera: Muscidea) is commonly regarded as an important

sanitary pest insect. However, due to its short
life cycle, high fecundity, and efficient digestion
of organic waste, it has also become a source of
protein, chitin and chitosan (Onifade et al. 2001;
Jing et al. 2007; Hao et al. 2008). Housefly larvae
could be used as a high protein livestock feed and

could thereby improve human nutrition by increasing the protein content of meat, milk, butter, and
eggs (Boushy 1991). The simplicity and low cost
of rearing housefly larvae on a commercial scale
has made this activity very popular in China. The
most feasible and easiest commercial utilisation of
housefly larvae is to raise them on poultry manure
and other organic wastes and then feed them fresh

Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, Grants No. KSCX2-YW-G-040
and No. KSCX2-YW-N-081.

616

Czech J. Food Sci.

to poultry or other livestock. In addition to their
value as a livestock feed, other valuable materials,
such as protein or peptides, chitosan, phospholipid,
and antibiotics have been extracted from housefly
larvae (Bridges & Price 1970; Iaboni et al. 1998;
Hou et al. 2007; Ai et al. 2008). Our preliminary
observations indicate that dried housefly larvae
contain approximately 55% protein, 9.1% crude
chitin, and 8.8% fat (unpublished data). We were
motivated to develop better techniques for rearing
and utilising housefly larvae because we had previously reared lepidopteran insects on an artificial
diet, much of which was wasted. Housefly larvae
provide a potential means of converting this waste
into useful biological products. A series of attempts
were carried out to isolate and characterise protein, chitin, fat, and other materials from housefly
larvae reared on the above-mentioned diet. This
paper describes the results of the experiments
undertaken to characterise chitosan extracted
from the cuticle of these larvae.
Chitin (-(1,4)-2-acetamido-2-deoxy-d-glucose)
is the second most abundant natural carbohydrate
polymer. Chitin and its derivative chitosan are
thought to have more than two hundred uses in
areas as diverse as agriculture, biomedicine, cosmetics, food, textiles, as well as the potential for
use as chelating agent and in refining industrial
effluents (Bahmani et al. 2000; Ravi Kumar 2000;
Rinaudo 2006; Aranaz et al. 2009). Currently, the
most available source of chitin is the exoskeletons
of crustaceans, particularly shrimps and crabs
(Minke & Blackwell 1978; Acosta et al. 1993).
However, the relatively high calcium, wax, and
pigment contents of shrimp and crab exoskeletons
cause the extraction of chitosan from them to be
relatively expensive. Compared to shrimps or crabs
(Rdde et al. 2008; Youn et al. 2009), the cuticle of
the housefly larvae is much easier to extract chitin
from because it contains smaller amounts of crude
protein, crude fat, and ash (our unpublished data).
However, no one has yet developed a method to
obtain high-grade chitosan from housefly larval
cuticle. In this paper, we describe the results of
the experiments aimed at optimising the extraction of chitosan from housefly larvae.
Material and Methods
Chitin extraction. Four-day old housefly larvae
were isolated from waste feed, rinsed and boiled

Vol. 29, 2011, No. 6: 616623

for 1015 min, and subsequently macerated in a
home juicer. The resultant granules of crude cuticle were strained from the mixture with a mesh
sieve, extensively washed, and freeze-dried. Protein
and lipid in the dry cuticle were removed by the
treatment with 1 mol/l NaOH solution (Hengye
Zhongyuan Chemical Co., Beijing, China) at 100C
for 3 hours. Crude chitin was obtained by rinsing
the mixture with water until reaching neutral pH
filtered with mesh sieve to remove water and then
freeze-dried.
Chitosan preparation. Several single-factor
experiments were conducted to evaluate various
reaction conditions for the deacetylation of chitin.
The factors were the granularity of the raw chitin, NaOH concentration, reaction temperature,
proportion of solution to material (PSM), reaction
time, and steeping time. Four grams of raw chitin
were used in each experiment. The experiments
were designed so that when one factor was tested
at different levels, all the other factors were fixed.
Other experimental constants included the diameter of the raw chitin granules (ca. 0.085mm),
NaOH concentration (50% w/v), reaction temperature (125C), PSM (30 ml/g), reaction time
(4 h), and steeping time (0.5 day). Based on these
single-factor experiments, an orthogonal experimental design was applied to identify the optimal
deacetylation conditions (Table 1). The data from
the orthogonal experiment were analysed by general
linear model analysis of variance with Duncans
multiple range test using SPSS software version
16.0 (SPSS Inc., Chicago, USA).
Removal of impurities from raw chitosan. To
avoid the negative impact of the impurities on
chitosan quality, a refinement procedure was developed for their removal from the deacetylated
product. After deacetylation, chitosan was filtered
and rinsed until neutral pH was achieved. Chitosan
was then dissolved in acetic acid solution (1%) and
the impurities were removed by filtration through
nylon net with a mesh diameter of 0.050 mm. The
Table 1. Orthogonal experimental combination of different
factors at various levels
Parameters

Levels
1

Proportion of solution
to material (ml/g)

22.5

25.0

27.5

Reaction temperature (C)

130

125

120

Reaction time (h)

617

Vol. 29, 2011, No. 6: 616623

pellucid filtrate was neutralised and precipitated
with NaOH solution until reaching neutral pH.
The precipitate was rinsed several times and then
freeze-dried after which the yield, i.e. the amount
of pure chitosan (% m/m) obtained from crude
chitin, was calculated.
Determination of the degree of deacetylation,
viscosity and ash content. The degree of deacetylation (DDA) was measured by the acid-base titration
method (Domard & Rinaudo 1983) with modifications. In brief, chitosan (0.1g) was dissolved
in 30 ml HCl aqueous solution (0.1 mol/l) at room
temperature with 56 drops of methyl orange
added. The red chitosan solution was titrated with
0.1 mol/l NaOH solution until it turned orange.
The DDA was calculated by the formula:
DDA (%) =

(C1V1 C2V2)
M 0.0994

0.016

where:
C1 concentration of standard HCl aqueous solution
(mol/l)
C2 standard NaOH solution (mol/l)
V1 volume of the standard HCl aqueous solution used
to dissolve chitosan (ml)
V2 volume of standard NaOH solution consumed
during titration (ml)
M weight of chitosan (g)

The number 0.016 (g) is the equivalent weight

of NH 2 group in 1 ml of standard 1 mol/l HCl
aqueous solution, and 0.0994 is the proportion
of NH 2 group by weight in chitosan.
One gram of the end product was dissolved in
1% acetic acid solution (100 ml) and its viscosity
was measured by means of NDJ-1 rotation viscometer (Hengping Instrument Co., Shanghai,
China) at 25C. The ash content was determined
gravimetrically after the incineration of the sample (0.51.0 g) in a muffle furnace at 500C for at
least 4 hours.
Chitosan FTIR analysis. Fourier transform infrared (FTIR) spectrum of the end product samples
was measured in KBr pellets in the transmission
mode in the range of 4004000 cm1 using a Bruker
Equinox 55 spectrophotometer (Bruker Optik,
Ettlingen, Germany).
X-ray diffraction analysis. The X-ray diffraction
(XRD) experiment was performed in the range
of 360 (2) using a PANalytical XPert PRO Xray diffractometer (PANalytical Co., Almelo, the
Netherlands).
618

Czech J. Food Sci.

Thermal analysis. Thermo-gravimetric analysis (TGA) and differential scanning calorimetry
(DSC) were carried out using Netzsch STA 499C
thermal analyser (NETZSCH-Gertebau GmbH.
Selb, Germany) at a heating rate of 10C/min under
nitrogen atmosphere.
Determination of physical and chemical properties and sanitary indices. Some physical and
chemical properties of the chitosan prepared under
the determined optimal processing conditions were
tested, including the amounts of water, ash, and
heavy metals, such as lead (Pb) and arsenic (As), the
DDA and viscosity, and bacteriological detections.
Lead (Pb) content was determined by graphite
furnace atomic absorption spectrometry according to the method of Oliveira et al. (2005) with
slight modifications. Arsenic (As) was measured
by hydride generation atomic fluorescence spectrometry according to the method of El-Hadri et
al. (2007). Aerobic bacterial count was determined
using the method described by Jarvis et al. (1977)
and coliforms were enumerated by the method of
Feldsine et al. (1994). Pathogens, including Staphylococcus aureus and Salmonella, were screened
by the methods of Bennett et al. (1986) and June
et al. (1995). The data were compared with those
of Chinese Fishery Trade Standard SC/T3403-2004
(Ministry of Agriculture of the Peoples Republic
of China 2005).
Results and Discussion
Chitosan preparation
The effects of variation of different factors on the
DDA, viscosity, yield, and ash content of the end
product are shown in Figures 1 and 2. As shown
in Figure 1, DDA generally increased with the
granularity of the raw chitin, NaOH concentration, reaction temperature, PSM, reaction time,
and steeping time, while viscosity was negatively
correlated with these factors. NaOH concentrations of below 50% resulted in significantly reduced
yields (Figure 2B). None of the factors examined
had a significant effect on ash content (Figure 2).
These results suggest that optimal conditions for
the preparation of chitosan from housefly larvae
were granularity of the raw chitin particles of about
0.85 mm, steeping time 1 day, reaction time of
48 h, reaction temperature of 120130C, and
PSM of 2030 ml/g.

Czech J. Food Sci.

300

81
80
79
0.60

0.425

82
240
78

270

74

240

70

0.30

Chitinsamplegranularity(mm)

(C)

300

(D)

180

50

55

120

60

NaOH concentration (%)

450

80

600

82

390

60

450

74

330

40

300

66

270

20

150

58

210

50

120

130

135

140

(F)

85

320

80

240

75

160

70

80

78

65

76

6
8
10
Reactiontime(h)

12

DDA(%)

400

20

25

30

35

40

150

350

86

90

15

Proportion of solution to material (ml/g)

Reactiontemperature(C)

(mPa.s)

DDA(%)

(E)

125

DDA (%)

90

(mPa.s)

750

DDA(%)

100

(mPa.s)

82

0.85

360

viscosity

(mPas)

330

2.0

D.D.

86

83
DDA(%)

(B) 90

360

viscosity

84

335

82

320

80

(mPa.s)

DDA

DDA (%)

84

(mPa.s)

(A)

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305
290
0.5

1.0

1.5

2.0

2.5

3.0

Steepingtime(day)

Figure 1. Effect of different factors on the degree of deacetylation (DDA) and viscosity of chitosan prepared from
housefly larvae. The DDA () and viscosity () were measured by the modified acid-base titration method and NDJ-1
viscosimetry, respectively, under different conditions, including chitin sample granularity (A), NaOH concentration
(B), reaction temperature (C), proportion of solution to material (D), reaction time (E) and steeping time (F)

Different factors had different effects on the

deacetylation reaction. Using exoskeleton particles
that were too fine resulted in a great decrease of the
yield and viscosity of the end product, while those
that were too coarse reduced DDA (Figures1A and
2A). Although NaOH concentrations below 50%
reduced the yield by inhibiting the deacetylation reaction (Figure 2B), excessive NaOH reduced viscosity
(Figure 1B). A low DDA and yield also occurred at
high ash content at 120C while high temperatures
caused a sharp decrease in viscosity and yield (Fig

ures 1C and 2C). Peak yields were obtained when

PSM was 25 ml/g or 30ml/g (Figure2D). Although
DDA increased with PSM, viscosity generally decreased (Figure 1D). A PSM of 15 ml/g seemed
unsuitable because of inadequate soaking of the
raw chitin resulting in a low DDA, viscosity, and a
high ash content in the end product (Figures 1D and
2D). Prolongation of the reaction or steeping time
improved DDA (Figures 1E and 1F), but excessive
reaction or steeping time decreased the yield and
viscosity (Figures 1E, 1F, 2E and 2F).
619

(B)

100

0.5

ash content

72

0.4

68

0.3

64

0.2

60

0.1

56
0.60

0.425

0.4

60

0.3

40

0.2

20

0.1

0.30

40

45

50

55

60

NaOH concentration (%)

Chitin sample granularity (mm)

0.5

72

0.4

72

0.4

64

0.3

64

0.3

56

0.2

56

0.2

48

0.1

48

0.1

40

40
120

125

130

135

Yield (%)

(D) 80
Ash content (%)

0.5

Yield (%)

(C) 80

140

70

0.40

65

0.30

60

0.20

55

0.10

10

12

(F)

25

30

35

40

0.00

Reaction time (h)

72

0.5
0.4

69
Yield (%)

0.50

Ash content (%)

Yield (%)

75

50

20

Proportion of solution to material (ml/g)

Reaction temperature (C)

(E)

15

Ash content (%)

0.85

80

0
2.0

0.5

ash content

yield

0.3
66
0.2
63
60

Ash content (%)

yield

Yield (%)

76

Ash content (%)

Yield (%)

(A)

Czech J. Food Sci.

Ash content (%)

Vol. 29, 2011, No. 6: 616623

0.1

0.5

1.0

1.5

2.0

2.5

3.0

Steeping time (days)

Figure 2. Effect of different factors on the yield and ash content of chitosan obtained from housefly larvae. The yield
() and ash content () were determined by standardised methods under different conditions, including chitin sample
granularity (A), NaOH concentration (B), reaction temperature (C), proportion of solution to material (D), reaction
time (E) and steeping time (F)

Optimal preparation conditions for chitosan

DDA, yield, viscosity, and ash content are the
four main parameters influencing the preparation efficiency and chitosan quality. Our singlefactor experiments (Figures 1 and 2) indicated
that DDA and yield were the two key parameters
that should be considered in the orthogonal experiment. Figure3 shows the results of the orthogonal experiment on the effects of various
620

levels of different factors on DDA and yield. PSM

had no significant effect on DDA or yield. The
reaction temperature and reaction time were the
key factors affecting the DDA, while the yield
was significantly affected only by the reaction
temperature. Taking into account the production costs, these results suggest that optimal
conditions for chitosan preparation are PSM of
22.5 ml/g, a reaction temperature of 125C, and
a reaction time of 6 hours.

Vol. 29, 2011, No. 6: 616623

90

90

80

80

70

70

60

60
DDA

50
40

A1 A2 A3

yield

B1 B2 B3

Yield (%)

100

DDA (%)

100

Trasmitance (%)

Czech J. Food Sci.

50
C1 C2 C3

4000 3500

40

3000

2500
2000 1500
Wave number (cm1)

1000

500

Figure 3. Results of an orthogonal experiment on the effect of varying proportion of solution to material (PSM),
reaction temperature, and reaction time, on degree of
deacetylation (DDA) and yield. Different letters in the
same curve indicate a significant difference (P < 0.05),
while the same letters in the same curve indicate no significant difference (P > 0.05)

Figure 4. Fourier transform infrared (FTIR) spectrum

of chitosan from housefly larvae produced under optimised preparation conditions (see text for explanation).
The FTIR spectrum was measured in KBr pellets in the
transmission mode in the range of 4004000 cm1 using
an EQUINOX 55 spectrophotometer

Characteristics of the chitosan prepared

responded to amide I band vibrations. The intensive

band at 15971600 cm1 corresponded to in-plane
bending vibration of NH2, a structural feature of
chitosan and the occurrence of deacetylation. The
band at 1355 cm1 was attributed to the stretching
of amide III vibration. The band at 1085 cm1 could
be attributed to the stretching of hydroxyl groups
of C 3-OH, and the band at 1039 cm 1 could correspond to the stretching of hydroxyl groups of
C6-OH. The band at 898cm1 corresponded to the
characteristic skeletal vibration of -anomers.
Usually, both chitin and chitosan from the exoskeletons of shrimps and crabs have strong re-

80

4
6
8

60
40

10

20
0
0

15

21

27
33 39
45
Wave number (cm1)

51

57

Figure 5. X-ray diffraction (XRD) patterns of chitosan from

housefly larvae produced under the optimised preparation
conditions (see text for explanation). The XRD experiment
was performed in the range of 360 (2) using an XPert
PRO X-ray diffractometer

12
14
16
18
100 200 300 400 500 600 700 800 900
Temperature (C)

DSC (mW/mg)

2
0
2

100
TGA residue mass (%)

Intensity

As shown in Figure 4, the FTIR spectrum of chitosan produced from housefly larvae was similar
to that of chitosan made from the exoskeletons of
shrimps, crabs, and other crustaceans; although
there were some differences in minor peaks, the
positions of the characteristic peaks and their intensities were nearly the same as those reported by
other researchers (Duarte et al. 2002; Pawlak &
Mucha 2003; Paulino et al. 2006; Abdou et al.
2008). The wide band at 3400 cm 1 corresponded
to OH stretching vibrations of water and hydroxyls,
and NH stretching vibrations of free amino groups.
The band observed at 2881 cm 1 corresponded to
CH stretching vibrations. The band at 1657 cm1 cor-

Figure 6. Curves of thermo-gravimetric analysis (TGA)

and differential scanning calorimetry (DSC) of chitosan
obtained from housefly larvae prepared under the optimised preparation conditions (see text for explanation).
TGA and DSC were carried out using Netzsch STA 499C
TG-DSC integrated instruments at the heating rate of
10C/min under a nitrogen atmosphere

621

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Czech J. Food Sci.

Table 2. Comparison of the physical and chemical properties and sanitary indices of the prepared chitosan with that
of Chinese Fishery Trade Standard SC/T3403-2004
Properties
Color
Appearance

Prepared chitosan

Industrial grade*

Food grade*

white

white or buff

white or buff

powder

powder or sheet

powder or sheet

Granularity (mm)

0.245

pH

6.96

6.5-8.5

6.58.5

DDA (%)

83.1

Viscosity (mPas)

347

12

10

0.13

2.0

0.5

1.0

1.0

Pb (mg/kg)

1.63

10

As (mg/kg)

0.41

0.5

Total bacterial count (cpu/g)

n.d.

1000

Water (%)
Ash (%)
Undissolved particles (%)

E. coli (MPN 100/g)

n.d.

Pathogens

n.d.

Physical and chemical properties and sanitary indices of industrial grade and food grade chitosan as the Chinese Fishery
Trade Standard SC/T3403-2004; n.d. not detected

flections at 2 around 910 and 2 of 2021 in

their XRD spectrum (Abdou et al. 2008). However,
the crystal peak near 10 in the chitosan obtained
from the housefly larvae gradually disappeared
and the crystal peak near 20 became wider and
weaker (Figure 5). This suggests that the intramolecular hydrogen bonds in the end product had
dramatically decreased after the deacetylation
reaction, and that its molecular structure was in
an amorphous state.
In thermograms, an upward DSC curve indicates
an exothermic reaction and a downward curve an
endothermic reaction. Two decomposition steps
can be seen in the TGA curve with an endothermic
reaction in the DSC curve (Figure 6). The first step
occurred at about 32100C and corresponded
to the evaporation of water. The second emerged
around 270320C and can be attributed to the
degradation of the chitosan sample.
Physical and chemical properties
and sanitary indices of chitosan obtained
from housefly larvae
Table 2 shows the results of the tests of the
physical and chemical properties, as well as the
sanitary indices of the chitosan produced under
the above given optimal preparation conditions.
622

The results indicate that water, ash, Pb, and As

contents, as well as pH of the end product, met
Chinese Fishery Trade Standard SC/T3403-2004
for food-grade chitosan. No pathogens were detected using the statutory examination methods
of the above-mentioned Chinese national standards. Since China has currently only industrial
and food grade trade standards for chitosan, we
can not verify whither the end product is of sufficient quality for medical use until the respective
standard is issued.
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Received for publication March 26, 2010
Accepted after corrections December 30, 2010

Corresponding author:
Dr. Qi-Lian Qin and Dr. Yi-Jun Wu, Chinese Academy of Sciences, Institute of Zoology, State Key Laboratory
of Integrated Management of Pest Insects and Rodents, Beijing 100101, P.R. China
tel.: + 86 10 648 070 56 (Q.-L. Qin); + 86 10 648 072 51 (Y.-J. Wu), e-mail: qinql@ioz.ac.cn (Q.-L. Qin); wuyj@ioz.ac.cn (Y.-J. Wu)

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